Identification of human DNA helicase V with the far upstream element-binding protein.

The properties of human DNA helicase V (HDH V) were studied in greater detail following an improved purification procedure. From 450 g of cultured cells, <0.1 mg of pure protein was isolated. HDH V unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand in an ATP- and Mg(2+)-dependent fashion. The enzyme is not processive and can also unwind partial RNA-RNA duplexes such as HDH IV and HDH VIII. The M:(r) determined by SDS-PAGE (66 kDa) corresponds to that measured under native conditions, suggesting that HDH V exists as a monomer in the nucleus. Microsequencing of the purified HDH V shows that this enzyme is identical to the far upstream element-binding protein (FBP), a protein that stimulates the activity of the c-myc gene by binding specifically to the 'FUSE' DNA region localized upstream of its promoter. The sequence of HDH V/FBP contains RGG motifs like HDH IV/nucleolin, HDH VIII/G3BP as well as other human RNA and DNA helicases identified by other laboratories.

Human DNA helicase VIII: a DNA and RNA helicase corresponding to the G3BP protein, an element of the ras transduction pathway.

Human DNA helicase VIII (HDH VIII) was isolated in the course of a systematic study of the DNA unwinding enzymes present in human cells. From a HeLa cell nuclear extract a protein with an Mrof 68 kDa in SDS-PAGE was isolated, characterised and micro-sequenced. The enzyme shows ATP- and Mg2+-dependent activity is not stimulated by RPA, prefers partially unwound 3'-tailed substrates and moves along the bound strand in the 5' to 3' direction. HDH VIII can also unwind partial RNA/DNA and RNA/RNA duplexes. Microsequencing of the polypeptide showed that this enzyme corresponds to G3BP, an element of the Ras pathway which binds specifically to the GTPase-activating protein. HDH VIII/G3BP is analogous to the heterogeneous nuclear ribonucleoproteins and contains a sequence rich in RGG boxes similar to the C-terminal domain of HDH IV/nucleolin, another DNA and RNA helicase.

Functional properties of the separate subunits of human DNA helicase II/Ku autoantigen.

The Ku antigen consists of two subunits of 70 and 83 kDa and is endowed with both duplex DNA end-binding capacity and helicase activity (human DNA helicase II). HeLa Ku can be isolated from in vitro cultured human cells uniquely as a heterodimer, and the subunits can be separated by electrophoresis only under denaturing conditions. To dissect the molecular functions of the two subunits of the heterodimer, we have cloned and expressed their cDNAs separately in Escherichia coli. The two activities of Ku (DNA binding and unwinding) were reconstituted by mixing and refolding both subunits in equimolar amounts (Tuteja, N., Tuteja, R., Ochem, A., Taneja, P., Huang, N-W., Simoncsits, A., Susic, S., Rahman, K., Marusic, L., Chen, J., Zang, J., Wang, S., Pongor, S., and Falaschi, A. (1994) EMBO J. 13, 4991-5001). Renaturation of the separate subunits can be achieved in the presence of a synthetic solubilizing and stabilizing agent, dimethyl ethylammonium propane sulfonate (NDSB 195). The helicase activity of the Ku protein resides uniquely in the 70-kDa subunit, whereas the DNA end-binding activity can be reconstituted only through renaturation of the two subunits in the heterodimeric form and is practically absent in the separate subunits. The 83-kDa subunit, when refolded in the absence of the 70-kDa subunit, forms homodimers unable to unwind DNA and bind duplex ends. The three separate species (heterodimer, 70-kDa subunit, and 83-kDa subunit homodimer) all have ssDNA-dependent ATPase activity.

Inhibition of DNA unwinding and ATPase activities of human DNA helicase II by chemotherapeutic agents.

DNA helicases catalyze the unwinding of duplex DNA and thus play important roles in the processing of DNA, little is known about the effects of various cytotoxic or antitumor chemotherapeutic agents on purified human DNA helicases. We have determined the effect of actinomycin C1, VP-16, camptothecin, ethidium bromide, ellipticine, nogalamycin, novobiocin, genistein, m-AMSA, aphidicolin and daunorubicin on the enzymatic activities of purified human DNA helicase II which was identified as Ku autoantigen. Ku contains DNA helicase, ATPase and DNA end binding activities. Our data have shown that out of several chemotherapeutic agents tested ethidium bromide, actinomycin C1, daunorubicin and nogalamycin were inhibitors of DNA unwinding activity of human DNA helicase II with ID50 values of 8.44 microM, 11.68 microM, 6.23 microM and 0.42 microM respectively. These inhibitors also inhibited the ATPase activity but not the DNA binding activity of this helicase. This inhibition could be due to binding of these drugs to DNA, thereby impeding the movement of the helicase for unwinding action which may be their most important pharmacological function against cancer cells.

Purification and properties of human DNA helicase VI.

A novel ATP-dependent DNA unwinding enzyme, called human DNA helicase VI (HDH VI), was purified to apparent homogeneity from HeLa cells and characterized. From 327 g of cultured cells, 0.44 mg of pure enzyme was recovered, free of DNA polymerase, ligase, topoisomerase, nicking and nuclease activities. The enzyme behaves as a monomer having an M(r) of 128 kDa, whether determined with SDS-PAGE, or in native conditions. Photoaffinity labelling with [alpha-32P]ATP labelled the 128 kDa protein. Only ATP or dATP hydrolysis supports the unwinding activity for which a divalent cation (Mg2+ > Mn2+) is required. HDH VI unwinds exclusively DNA duplexes with an annealed portion < 32 bp and prefers a replication fork-like structure of the substrate. It cannot unwind blunt-end duplexes and is inactive also on DNA-RNA or RNA-RNA hybrids. HDH VI unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand.

Human DNA helicase II: a novel DNA unwinding enzyme identified as the Ku autoantigen.

Human DNA helicase II (HDH II) is a novel ATP-dependent DNA unwinding enzyme, purified to apparent homogeneity from HeLa cells, which (i) unwinds exclusively DNA duplexes, (ii) prefers partially unwound substrates and (iii) proceeds in the 3' to 5' direction on the bound strand. HDH II is a heterodimer of 72 and 87 kDa polypeptides. It shows single-stranded DNA-dependent ATPase activity, as well as double-stranded DNA binding capacity. All these activities comigrate in gel filtration and glycerol gradients, giving a sedimentation coefficient of 7.4S and a Stokes radius of approximately 46 A, corresponding to a native molecular weight of 158 kDa. The antibodies raised in rabbit against either polypeptide can remove from the solution all the activities of HDH II. Photoaffinity labelling with [alpha-32P]ATP labelled both polypeptides. Microsequencing of the separate polypeptides of HDH II and cross-reaction with specific antibodies showed that this enzyme is identical to Ku, an autoantigen recognized by the sera of scleroderma and lupus erythematosus patients, which binds specifically to duplex DNA ends and is regulator of a DNA-dependent protein kinase. Recombinant HDH II/Ku protein expressed in and purified from Escherichia coli cells showed DNA binding and helicase activities indistinguishable from those of the isolated protein. The exclusively nuclear location of HDH II/Ku antigen, its highly specific affinity for double-stranded DNA, its abundance and its newly demonstrated ability to unwind exclusively DNA duplexes, point to an additional, if still unclear, role for this molecule in DNA metabolism.

Interactions of USF and Ku antigen with a human DNA region containing a replication origin.

By means of a combination of ion-exchange and sequence-specific affinity chromatography techniques, we have purified to homogeneity two protein complexes binding in a human DNA region (B48) previously recognized to contain a DNA replication origin. The DNA sequence used for the protein purification (B48 binding site) contains a binding site for basic-helix-loop-helix DNA binding proteins. The first complex is composed of two polypeptides of 42- and 44-kDa; its size, heat stability, and target DNA sequence suggest that it corresponds to transcription factor USF; furthermore, the 42-kDa polypeptide is recognized by antibodies raised against 43-kDa-USF. The second complex is represented by equimolar amounts of two proteins of 72 and 87 kDa; microsequencing of the two species indicated that they correspond to the human Ku antigen. In analogy with Ku, they produce a regular pattern of footprints without an apparent sequence-specificity, and their binding can be competed by unspecific DNA provided that it contains free ends. The potential role of B48 binding site and of these cognate proteins in origin activation is discussed.

DNA helicase III from HeLa cells: an enzyme that acts preferentially on partially unwound DNA duplexes.

Human DNA helicase III, a novel DNA unwinding enzyme, has been purified to apparent homogeneity from nuclear extracts of HeLa cells and characterized. The activity was measured by using a strand displacement assay with a 32P labeled oligonucleotide annealed to M13 ssDNA. From 305 grams of cultured cells 0.26 mg of pure protein was isolated which was free of DNA topoisomerase, ligase, nicking and nuclease activities. The apparent molecular weight is 46 kDa on SDS polyacrylamide gel electrophoresis. The enzyme shows also DNA dependent ATPase activity and moves unidirectionally along the bound strand in 3' to 5' direction. It prefers ATP to dATP as a cofactor and requires a divalent cation (Mg2+ > Mn2+). Helicase III cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids and requires more than 84 bases of ssDNA in order to exert its unwinding activity. This enzyme is unique among human helicases as it requires a fork-like structure on the substrate for maximum activity, contrary to the previously described human DNA helicases I and IV, (Tuteja et al. Nucleic Acids Res. 18, 6785-6792, 1990; Tuteja et al. Nucleic Acids Res. 19, 3613-3618, 1991).